RNA molecules can be designed to form a wide variety of compact and thermodynamically stable three-dimensional structures (nanoparticles or NP) suitable for a broad range of nanotechnological and biomedical applications. Currently, two main strategies are used to design various RNA NP. In one strategy, based on the principles of RNA architectonics the single-stranded RNAs (ssRNAs) form monomers with pre-folded structural motifs followed by bottom-up assemblies via intermolecular tertiary hydrogen bond formations (e.g. hexameric nanorings). In another strategy, ssRNAs are designed to avoid stable internal secondary structures, and their assemblies are built solely from intermolecular interactions independent of any tertiary bindings (e.g. nanocubes). Fusion of the individual RNAs participating in NP formation with functional/therapeutic RNAs (ribozymes, small interfering RNA (siRNAs), aptamers, etc) allows precise control over the stoichiometric organization and simultaneous delivery of different RNA functionalities into cells. However, biomedical integration of such functional RNA NP is somewhat limited by at least three obstacles:
(i) The Cost and Size Limitations Associated with Chemical Synthesis of RNA.
The addition of functional groups to RNA scaffolds typically involves increasing the length of the individual RNA strands entering into the composition of the RNA nanoparticles. This strategy often requires the synthesis of RNAs in lengths that exceed what is currently available by commercial synthesis (i.e. RNAs that are greater than 60-nt). Furthermore, because chemical synthesis of RNA is relatively expensive compared to DNA, long RNAs are usually prepared by in vitro transcription with bacteriophage T7 RNA polymerase.
(ii) The Complexity of RNA NP Production.
Given that the synthesis of DNA oligos are relatively inexpensive, RNA synthesis usually relies on the use of DNA templates coding individual strands of RNA NP which are transcribed in vitro. Resulting RNAs are gel purified, recovered from purification and combined at equimolar quantities. Thermal denaturation and specific refolding protocols are used to ensure the desired NP formation. Denaturation and refolding conditions strongly depend on NP design strategies as well as the sequences of individual RNAs. Therefore, each NP requires an optimization of the assembly protocol.
(iii) Low Retention Time of RNA NP in the Patient Blood Stream Due to their Susceptibility to Nuclease Degradation.
Inclusion of dNMPs chemically modified at the 2′-position of the ribose sugar into the RNA strands of RNA NPs offers a promising way to increase the retention time of functional RNA NPs in the blood stream. Production of chemically modified RNA NPs has been previously achieved through transcription of individual RNA strands by mutant bacteriophage T7 RNA polymerase in the presence of 2′-fluorinated dUTP, and unmodified ATP, GTP, and CTP. This Y639F mutant enzyme (defective in discrimination between rNTP and dNTP substrates) is commercially available and relatively expensive. The purified RNA strands were used for NP assemblies by thermal denaturation and refolding. Notably, the overall yields of fluorinated RNAs, produced by the mutant polymerase according to the manufacturer's protocol, are significantly lower than the amount of unmodified transcripts (data not shown), precluding the large-scale production of chemically modified NPs. Apparently, the enzyme mutation does not completely alleviate the inefficient incorporation of several modified residues in a row, which sometimes is essential for formation of the full-length transcript.
Accordingly, improved methods of producing RNA nanoparticles are needed